Monitoring of liquids for disease-associated materials

ABSTRACT

A method for monitoring liquids for the presence of disease-associated materials, so as to provide a non-invasive means for the detection of various materials associated with cancer, autoimmune, neuro-degenerative and other disorders. The method provided comprises contacting a sample of the liquid with a solid, non-buoyant particulate material having free ionic valencies so as to concentrate the disease-modified or associated proteins in the sample and then monitoring the resulting disease-modified or associated proteins concentrated on the particulate material.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a continuation in part ofinternational application PCT/GB98/00374 filed on Feb. 6, 1998 by thesame applicant as the present invention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the monitoring of liquids fordisease-associated materials and more specifically to the monitoring ofliquids for materials associated with autoimmune and other diseases, allusing non-invasive means.

[0003] At present, the principal methods for monitoring infectious andautoimmune disorders, cancer and the like, such as Alzheimer's disease,multiple sclerosis, spongiform encephalopathies etc. are invasivetechniques involving the monitoring of pathological changes insurgically accessible tissue. Similarly, principal methods formonitoring various cancers also involve invasive techniques. Amyloidplaques, for example, are a common neuropathological feature ofAlzheimer's disease and would conventionally require invasive surgery inorder to be detected, which is generally undesirable. These surgicalmethods are expensive and time consuming and are often only undertakenwhen a disease is at an advanced stage.

[0004] Spongiform encephalopathies, such as Creutzfeldt-Jakob disease(CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS) and Kuru in humans;scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) incattle, mink and cats are all transmissible (infective)neuro-degenerative disorders implicating vacuolation of neurons.

[0005] At present, the most reliable method of detecting anencephalopathy is histologically, especially by electron microscopy, butthis requires brain tissue removed following autopsy of the dead victim.Although neurological examination and electro-encephalographs (EEG) canprovide accurate diagnosis in many cases of encephalopathy, there is anurgent need for a definitive test during life, one which can detect thedisease during its early stages and which is non-intrusive.

[0006] Therefore, an accurate, non-invasive test would provide means toaid in the early detection and diagnosis of various disorders, therebyimproving the possibility for the early treatment of the disease, hencepotentially increasing the chances of combatting or arresting thedisorder.

[0007] The protein associated with for example the neuro-degenerativedisorder CJD is thought to be a particle termed a “nemavirus”. Incontrast to the morphology of a common virus, which has a two layerstructure of nucleic acid protected by an outer coat, the nemavirusparticle has an unusual three layer structure which comprises:

[0008] 1. a protein core,

[0009] 2. single stranded DNA, and

[0010] 3. a protein coat.

[0011] The single stranded DNA is sandwiched between the protein coreand the protein coat. Single stranded DNA from scrapie has been partlysequenced and contains a palindromic repeat sequence TACGTA. Thescrapie-specific nucleic acid is single stranded DNA and includes thesequence (TACGTA)_(n) where n is at least 6. The basic six unit of thisrepeat sequence is palindromic, in the sense that a complementary DNAwould have the same TACGTA sequence when read in the 5′ to the 3′direction. The full length sequence of the DNA is not known, but it issuspected that n is very much larger than 6, perhaps of the order of 20to 30. Although the DNA sequence is scrapie-specific, BSE, scrapie, CJDand other encephalopathies are thought to result from the same proteinassociated with the neuro-degenerative disorder transferred to anotherspecies. It is therefore believed that the TACGTA palindromic sequenceappears in all known spongiform encephalopathies and possibly others.

[0012] The protein coat has not yet been characterised. The protein corecomprises the protease-resistant protein (PrP) which is termed a“prion”. A prion is encoded by a cellular gene of the host and isthought to contain little or no nucleic acid. However, the cellular formof the prion protein is modified into protease-resistant protein (PrP),by an accessory protein , “Nemo Corrupta” coded by single stranded DNA(PESM, 212, 208-224, (1996). This feature distinguishes prions sharplyfrom virions. To date, no prion-specific nucleic acid which is requiredfor transmission of disease has been identified.

[0013] Virus-like nemaviruses are tubulofilamentous particles in shape,typically 23-26 nm in diameter. They are consistently detected in thebrains of all known spongiform encephalopathies. These particles have acore of prion in a rod-like form; the prion rods being also termedscrapie-associated fibrils (SAF). Over the core is a layer of DNA,removable by DNAse; above the core is an outer protein coat which isdigestible by a protease.

[0014] It would be desirable to have a method of diagnosis based onnucleic acid identification or on the core structure of the nemavirusprotease-resistant protein in a living human or animal. Such methodshave been suggested where a probe of DNA derived from the gene sequencecoding for a prion protein are used. However, since it is well knownthat prion protein is encoded by a normal chromosomal gene found in allmammals, including those affected by encephalopathies, the above workhas not gained acceptance. PCT Patent Application WO89/11545 (Institutefor Animal Health Ltd) purports to describe a method of detection ofscrapie susceptibility by use of a restriction fragment lengthpolymorphism (RFLP) linked to the so called Sinc gene associated withshort incubation times of sheep infected by scrapie. The RFLP is said tobe located in a non-coding portion associated with the gene for theprion. At best, this method would detect only sheep with the shortincubation time characteristic. Hitherto, methods of diagnosis based onnucleic acid identification have not been very successful or are likelyto be unsuccessful, since an encephalopathy specific nucleic acid haseluded detection despite numerous attempts.

[0015] In human CJD cases, infectivity associated with theneuro-degenerative disorder has been consistently shown by titrationstudies to be present in blood. Although the protein associated with theneuro-degenerative disorder is present in urine of CJD cases, there isno known technique of diagnosis based on urine.

[0016] UK patent 2258867, describes a method for the diagnosis ofencephalopathy using animal tissue. This method includes the use of ascrapie-specific nucleic acid, part of which can be labelled and used asan oligonucleotide probe in a hybridisation assay. Alternately, asequence from the scrapie-specific nucleic acid is used as a primer in apolymerase chain reaction to make sufficient quantities to allowdetection by a restriction fragment length method.

OBJECTS OF THE INVENTION

[0017] It is an object of the present invention to provide a method formonitoring liquids for disease-associated materials, which can be usedfor detection of materials associated with diseases such as cancer,autoimmune and neuro-degenerative disorders.

[0018] It is a further object of the present invention to providenon-invasive means for the detection of various materials associatedwith cancer, and autoimmune and other disorders.

[0019] It is a further object of the present invention to provide meansfor the detection of materials associated with autoimmune and otherdisorders at an earlier stage than is possible using techniquescurrently available (particularly where the etiology is unknown ordifficult to determine).

SUMMARY OF THE INVENTION

[0020] According to a first aspect of the present invention, there isprovided a method of monitoring a liquid for the presence ofdisease-modified or associated proteins, comprising the steps of:

[0021] (a) contacting a sample of the liquid with a solid, non-buoyantparticulate material having free ionic valencies so as to concentratethe disease-modified or associated proteins in the sample; and

[0022] (b) monitoring the resulting disease-modified or associatedproteins concentrated on the particulate material.

[0023] The concentration of the disease-modified or associated proteinstakes place as a result of aggregation thereof on the surface of theparticulate material.

[0024] It is a preferred feature of the present invention that thesample of liquid body fluid comprises a urine sample or a sample ofanother body fluid (such as serum or cerebral spinal fluid) comprisingdetectable levels of a disease-modified protein or detectable levels ofviral matter.

[0025] According to the present invention, the disease-modified proteinis a protein or a fragment thereof which is modified due to a disease ina host body and which protein or fragment thereof is excreted as thedisease process begins. For example, it is known that amyloid β-proteinis derived from amyloid β-precursor protein which is encoded by a normalhost gene mapped to chromosome 21. In Alzheimer's disease, amyloidβ-precursor protein slices into 3 segments as the disease progresses,one of the segments, typically the middle segment, being amyloidβ-protein (a 4 KDa protein which forms plaques as seen in brain sectionsof Alzheimer's patients). The remaining two segments of the amyloidprecursor protein have not been demonstrated in brain tissue ofAlzheimer's patients. In patients testing positive for Alzheimer'sdisease, the presence of C-terminal segments of the amyloid β-precursorprotein, or other segments, may be shown. In contrast, the urine ofpatients testing negative for Alzheimer's disease will not containsegments of the amyloid β-precursor protein. Such protein modificationshave been found to occur in both infectious and non-infectious diseases,such as cancer.

[0026] According to the present invention, when the disorder isAlzheimer's disease, the disease-modified protein is typically amyloidβ-protein. Furthermore, when the disorder is multiple sclerosis, thedisease-modified protein is typically myelin. When the disorder is abovine spongiform encephalopathy or Creutzfeldt Jakob disease, thedisease-modified protein is typically protease-resistant protein.

[0027] According to the present invention, viruses such ascytomegalovirus, papillomavirus or the AIDS virus excreted in urine maybe detectable.

[0028] According to the present invention, the protein may be associatedwith neuro-degenerative disorder, such as a nemavirus which may beconcentrated from a sample of a body fluid, such as urine, taken fromthe animal.

[0029] According to a further preferred feature of the presentinvention, the disorder may be Alzheimer's disease, multiple sclerosisor a spongiform encephalopathy. Furthermore, since disease modifiedproteins have been demonstrated in cancer, for example in cancer of thecervix, the method according to the present invention may also beapplicable to the detection and subsequent diagnosis of various forms ofcancer. Similarly, various viruses associated with certain cancers,growths etc. have also been demonstrated in urine samples.

[0030] As indicated, the disease-modified or associated protein isconcentrated from a body fluid, such as urine, using a solid non-buoyantparticulate material, a preferred example of which is calcium phosphate.Calcium phosphate is widely used in transformation experiments to allowthe introduction of DNA into a living cell, wherein it causes theprecipitation of DNA. However, it has not been previously suggested forthe purpose of concentrating a disease-modified or associated protein ina diagnostic sample of urine or the like.

[0031] A further example of a solid non-buoyant particulate material ishydroxyapatite or the like. Hydroxyapatite and similar media, asion-exchange chromatography media, have previously been used to purifyand concentrate viruses and their related soluble antigens. Such mediahave had limited application in the clinical diagnosis of human andanimal diseases largely due to the impracticality of handling largesample volumes with slow transit of liquids through an ion-exchangecolumn. A further problem is the low concentration of disease-relatedproteins which are in competition with contaminating proteins forexchange sites on the particulate materials. The method according to thepresent invention overcomes some of these difficulties by use of amedium which discriminates adsorption of albumin (in other words,proteins such as albumin are selectively not complexed as the medium iscaused to lose the charge that allows albumin to complex).

[0032] The particulate material is preferably in the form of granules.Part of the disease-modified or associated protein, for example aprotein associated with neuro-degenerative disorder (in the case ofspongiform encephalopathies) or amyloid precursor protein APP (in thecase of a non-transmissible neuro-degenerative disease, such asAlzheimer's and basic myelin protein oligocyte for multiple sclerosis),is thought to bind to the surface of the granules.

[0033] The steps leading to the concentration of the disease-modified orassociated protein from a sample of body fluid such as urine typicallycomprise:

[0034] (a) collecting and centrifuging a sample of urine from aninfected animal;

[0035] (b) collecting the supernatant produced following centrifugationof the sample of urine;

[0036] (c) adding a buffer and a solid, non-buoyant particulate materialhaving free ionic valencies (such as calcium phosphate granules) to thesupernatant;

[0037] (d) centrifuging the resulting mixture of buffer, particulatematerial and supernatant;

[0038] (e) collecting particulate material following centrifugation;

[0039] (f) adding a buffer to the particulate material;

[0040] (g) centrifuging the mixture of buffer and particulate material;

[0041] (h) collecting the particulate material;

[0042] (i) adding a buffer to the particulate material;

[0043] (j) centrifuging a mixture of the buffer and the particulatematerial; and

[0044] (k) collecting the particulate material containing thedisease-modified or associated protein.

[0045] The sample of urine or the like can be concentrated 100 fold ormore using calcium phosphate or other non-buoyant particulate materialin the method according to the invention; the concentrated urine canthen be used in several ways to allow diagnosis of diseases such ascancer, autoimmune and neuro-degenerative disorders.

[0046] According to a further aspect of the present invention, there isprovided a method of monitoring a liquid for the presence ofdisease-modified or associated proteins, comprising the steps of:

[0047] (a) providing a sample of the liquid;

[0048] (b) passing the sample through a solid filter medium having freeionic valencies so as to complex at least one biological material to themedium, the biological material being selected from the group consistingof disease-modified or associated protein, a fragment thereof, a virusor a fragment thereof; and

[0049] (c) monitoring at least a part of the complexed biologicalmaterial, wherein the presence of at least a part of the biologicalmaterial is indicative of an association of the liquid with the relevantdisease.

[0050] According to the present invention, the filter medium preferablycomprises a sheet-like member with a pore size ranging from 1 to 100microns. The pore size of the filter may be varied according to the sizeof the particles to be entrapped. Furthermore, the filter preferablycomprises a gauze and/or cotton fiber.

[0051] In an alternate embodiment of the present invention, anon-buoyant particulate material having free ionic valencies(such ascalcium phosphate) may be used in addition to the filter medium.

[0052] According to one aspect of the present invention, theconcentrated or filtered sample of body fluid such as urine can be usedfor the detection of disease-modified or associated proteins usingelectron microscopy. In such a method, a grid is brought into contactwith the sample of concentrated or filtered urine or the like and thenthe grid is fixed and stained. For example, the tubulofilamentousparticles that are characteristic of the nemavirus associated withneuro-degenerative disorder may be visualized by electron microscopy.

[0053] Diagnosis can alternatively be carried out by means of, forexample, an enzyme-linked immunosorbent assay (ELISA). The ELISAtechnique can be automated to provide a semi-quantitative result. Thecalcium phosphate for the concentration of the disease-modified orassociated protein would be included as part of an ELISA kit. Such a kitaccording to the invention preferably further comprises a blockingbuffer, an antibody to the disease-modified or associated protein and anantibody conjugate. A kit according to the invention preferablycomprises:

[0054] (a) a solid, non-buoyant particulate material having free ionicvalencies (such as calcium phosphate) in a form capable of complexingwith protein present in a body fluid;

[0055] (b) a blocking buffer capable of complexing with any of theparticulate material not complexed with the protein;

[0056] (c) a first antibody material capable of complexing with thecomplexed protein; and

[0057] (d) a further antibody which is capable of complexing with thefirst antibody.

[0058] When the neuro-degenerative disorder is a spongiformencephalopathy, an antibody to PrP may be added which will bind to theprotein associated with the neuro-degenerative disorder on the surfaceof the particulate material. This is generally followed by a secondantibody which will bind to the previous antibody, the second antibodybeing conjugated to a marker enzyme to allow detection of the proteinassociated with the neuro-degenerative disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The use of calcium phosphate as an exemplary particulate materialin the concentration of the disease-modified or associated protein andthe subsequent detection using an ELISA method is shown schematically inFIGS. 1 to 6 of the accompanying drawings, which are by way of exampleonly. In the drawings:

[0060]FIG. 1 shows a reaction vessel 1, having therein an exemplarycalcium phosphate granule 2 and a disease-modified or associated protein3;

[0061]FIG. 2 shows the disease-modified or associated protein 3concentrated on the surface of the calcium phosphate granule 2;

[0062]FIG. 3 shows the unbonded sites on the surface of the calciumphosphate granule 2 blocked on the addition of blocking buffer (such asmilk) 4;

[0063]FIG. 4 shows the addition of a first antibody against thedisease-modified or associated protein 5;

[0064]FIG. 5 shows binding of the first antibody 5 to thedisease-modified or associated protein 3 which is still bonded to thesurface of the calcium phosphate granule 2;

[0065]FIG. 6 shows antibody detection using a second antibody 6conjugated to a marker enzyme such as horseradish peroxidase or alkalinephosphatase; and

[0066]FIG. 7 is a photograph of a stained blot obtained in an exemplarydiagnostic method according to the invention.

[0067] Another method for the diagnosis of diseases such as cancer,autoimmune and neuro-degenerative disorders from the concentrated orfiltered sample of body fluid such as urine is to amplify the DNA in thesample by using a polymerase chain reaction (PCR).

[0068] In a preferred method for the diagnosis of encephalopathy, thepalindromic oligonucleotide described above is used to amplify thesample DNA. Such oligonucleotides will not normally be longer than 200nucleotides, even when used as probes; generally, they are likely to bevery much shorter. Thus, for PCR purposes they are unlikely to comprisemore than 24 nucleotides of the palindrome, plus an optional 5′-end ortail of (say) 8 to 20 nucleotides, making 32 to 44 nucleotides in all.The PCR will yield a product in the form of DNA of varying lengthscontaining the palindromic sequence. This can preferably be analyzed bya method relying on restriction by an enzyme.

[0069] The PCR product will produce bands of various molecular weights.In some instances the encephalopathy-specific DNA will be primed nearits 3′-end, which will generate multiple copies of large molecules. ThePCR product may be divided into two portions, of which the first may berun on a resolving gel to show a band of high molecular weightassociated with the encephalopathy-specific DNA, the second portionbeing restricted with a restriction enzyme which cuts the palindromicsequence. This restriction will severely reduce the length of the longerDNA and eliminate certain other bands of shorter DNA altogether.Multiple restrictions of TACGTA will produce many bands of molecularweight too low to be detected. Restricted product can be compared withthe unrestricted product, whereby disappearance of longer lengths of DNAupon restriction indicates the presence of the encephalopathy-specificDNA in the sample.

[0070] Examples of suitable restriction enzymes are SnaBI and AccI,which cut between the C and G of TACGTA and Bst11071 which cuts betweenA and T of one TACGTA sequence and the next TACGTA sequence. Suchenzymes recognise the six-base sequence and leave blunt ends.

[0071] The sample of urine or other body fluid containing theconcentrated disease-modified or associated protein can be used in afurther assay for the diagnosis of diseases such as cancer, autoimmuneand neuro-degenerative disorders, using a hybridization method. In thehybridization method, the sample of urine or the like, containing thedisease-modified or associated protein, can be used as it is, orpreferably, it may be amplified before use, for example, using a PCRmethod. The hybridization probe is preferably from 16 to 100 nucleotideslong, especially about 40 nucleotides long. The hybridization assay canbe carried out in a conventional manner; Southern blotting is preferred.For use in a hybridization assay, the oligonucleotide will normally beused in a labelled form, labelling being by any appropriate method suchas radiolabelling, for example, by ³²P or ³⁵S, or by biotinylation(which can be followed by reaction with labelled avidin). However, it isalso possible to use an unlabelled oligonucleotide as a probe providedthat it is subsequently linked to a label. For example, theoligonucleotide could be provided with a poly-C tail which could belinked subsequently to labelled poly-G.

[0072] An alternative method for the diagnosis of diseases such ascancer, autoimmune and neuro-degenerative disorders is using a proteinblotting method (Western blotting) which comprises detecting the proteinof interest on the surface of a membrane (such as nitrocellulose) anddetection of the protein using antibody technology.

[0073] The present invention has been described with particularreference to purification and detection of protein and viral matter fromsamples of body fluid such as urine. According to a further embodimentof the present invention, the solid non-buoyant particulate material maybe used to concentrate viral samples form water, and/or the filtertechnology may be used to purify viral samples from water. The methodaccording to the invention may prove useful in the detection of viraland/or bacterial matter from sea water, swimming pool water, tap wateror the like.

EXAMPLES Purification of a Disease-Modified or Associated Protein from aSample (for Example Urine)

[0074] A sample of urine was collected from an animal suspected ofhaving neuro-degenerative disorder. The urine sample was centrifuged andthe supernatant collected. A suitable buffer and calcium phosphategranules were then added to the supernatant. This mixture of urinesupernatant, buffer and calcium phosphate was allowed to rest at roomtemperature (with regular mixing) for at least ten minutes. The mixturewas then centrifuged and the calcium phosphate granules collected. Asuitable buffer was then added to the calcium phosphate granulesfollowed by a further centrifugation step. The calcium phosphategranules were collected and the above addition of buffer andcentrifugation step was repeated a further two times. The calciumphosphate granules were collected for the detection of a possibleprotein associated with a neuro-degenerative disorder using any ofexamples A, B, C, D, E or F detailed below.

Example A Enzyme Linked Immunosorbent Assay

[0075] The calcium phosphate granules obtained following the abovepurification stage were used.

[0076] A suitable blocking buffer (for example milk) was added to thecalcium phosphate granules and the solution was left mixing for at leastsixty minutes. The solution was then centrifuged and the supernatantdiscarded. To the calcium phosphate granules that remain some phosphatebuffered saline (PBS) containing Tween 20 was added and this wasfollowed by a further centrifugation step. The above PBS-Tween 20 washstep was repeated at least four times. A first antibody was then addedto the calcium phosphate granules. This was left to stand for at least60 minutes with mixing at regular intervals. PBS-Tween 20 was added andfollowed by a centrifugation step. The supernatant was discarded and thePBS-Tween 20 wash step repeated at least four times. A second antibody,(one conjugated to a marker enzyme) was then added to the calciumphosphate granules and left mixing for at least sixty minutes. PBS-Tween20 was then added followed by a centrifugation step. The supernatant wasdiscarded and the wash step repeated with PBS-Tween 20 at least fourtimes.

[0077] A substrate suitable for detection of the marker enzyme on thesecond antibody was then added. This was left to stand for at leasttwenty minutes and the reaction stopped by addition of a suitablereagent, such as concentrated sulfuric acid. Following centrifugation,the supernatant was collected and read photometrically at a suitablewavelength.

Example B Preparation of Grids for Electron Microscopy

[0078] The calcium phosphate granules obtained following thepurification stage were used.

[0079] Ethylenediaminetetraacetic acid (EDTA) was added to the calciumphosphate granules and mixed until a clear solution was produced. Acarbon-coated grid was lowered into tubes containing some distilledwater making sure the carbon/Formvar film was facing upwards. For eachspecimen at least two grids were prepared using the clear solution whichwas then transferred into the tube whilst gently rinsing off thedistilled water. The grids were then centrifuged horizontally. After thecentrifugation step sodium dodecyl sulfate was added and the gridstransferred into distilled water. The grids were then washed severaltimes in distilled water. The water was then momentarily replaced withglutaraldehyde containing ruthenium red. This solution was then rinsedout with distilled water and the grids were then momentarily introducedto a solution of osmic acid containing ruthenium red. The grids wereagain rinsed several times with distilled water. After the final wash ofwater with a drop of phosphotungstic acid the grids were dried andexamined under an electron microscope.

Example C Polymerase Chain Reaction

[0080] Again the calcium phosphate granules obtained following thepurification stage were used.

[0081] EDTA was added to the calcium phosphate granules until a clearsolution was produced. Some of this clear solution was taken andincubated with proteinase K for at least one hour at 55° C. Theproteinase K was then heat inactivated at 95° C. and the solution usedas a template in a polymerase chain reaction (PCR) A dNTP mix, primers,a buffer and AmpliTaq DNA polymerase were then added to the reactionmixture. Thirty cycles of PCR were carried out comprising a denaturationstage, annealing of primers and an extension stage. The PCR product wasthen cut with the restriction enzyme SnaB1. Cut and uncut PCR productwas then analyzed using electrophoresis and the fragments visualized onthe agarose gel after staining with ethidium bromide.

Example D Protein Blotting for Immunoassay

[0082] Bio-Dot apparatus was used for the immunoblotting procedure.Nitrocellulose membranes were pre-wetted in Tris saline buffer (TSB)prior to placing in the bio-dot apparatus. After re-hydrating themembrane the wells of the apparatus were filled with antigen. Theantigen solution being the clear solution produced on mixing the calciumphosphate granules (obtained from the purification stage) with EDTA. Theentire antigen sample was allowed to filter through the membrane. Afterthe antigen samples had completely drained from the apparatus the Trissaline buffer (TSB) was added and the liquid allowed to filter through.Blocking solution was then added to each well and the liquid allowed tofilter through the apparatus. Tween-tris saline buffer (TTSB) washsolution was added to the apparatus and the flow valve adjusted toproduce a vacuum to pull the wash solution through the membrane. Thevacuum was then disconnected and a first antibody solution added to eachsample well. The solution was allowed to filter through the membrane andthe vacuum was re-applied to remove any excess liquid from the samplewells. TTSB wash solution was then added and pulled through the membranewith the aid of a vacuum. The wash process was then repeated threetimes.

[0083] Conjugated antibody solution was added to each well and theliquid allowed to filter through. TTSB wash solution was then added toeach well and the solution pulled through the membrane with the aid of avacuum. This wash process was repeated twice. The membrane was removedand placed in the colour development vessel. The membrane was thenremoved and washed with TSB to remove excess Tween 20. The membrane wasthen incubated in a suitable substrate until the development of spotswere seen. After this time the membrane was rinsed in distilled waterand photographed.

Example E Southern Blotting

[0084] Again the calcium phosphate granules obtained following thepurification stage were used.

[0085] Concentrated NaOH and DMSO was added to the calcium phosphategranules. The solution was mixed and heated and then cooled down to roomtemperature after which concentrated ammonium acetate was added.Nitrocellulose membrane was then wetted in 6XSSC and the bio-dotapparatus assembled. The DNA sample was applied and allowed to filterthrough the membrane. After the sample had filtered 2XSSC was added toeach well and vacuum applied to remove the liquid. The blot membrane wasremoved and washed in 2XSSC. The nitrocellulose membrane was then bakedunder vacuum before hydridization.

Example F Western Blotting

[0086] The calcium phosphate granules obtained following thepurification steps outlined were used.

[0087] Sodium dodecyl sulfate containing proteinase k was then added tothe calcium phosphate granules and the mixture incubated for at least 60minutes at 55° C. β-mercaptoethanol was added after the incubationperiod and the mixture was then boiled. Following this polyacrylamidegel electrophoresis was carried out. Proteins on the polyacrylamide gelwere then transferred to a nitrocellulose membrane. The membrane was airdried and then washed in tris buffered saline. Any unabsorbed sites werethen blocked using heat inactivated horse serum and goat milk. A firstantibody made up in tris-buffered saline containing Tween 20 and milkwas then applied to the membrane which was left to incubate for at leastone hour. The membrane was then washed several times. A second antibodyconjugated to a marker enzyme (which was also made up in a solution oftris-buffered saline containing Tween 20) was then applied to themembrane. This was left to incubate for at least 60 minutes and thenwashed in a solution of tris buffered saline to remove excess Tween 20.The membrane was then incubated in a suitable substrate until thedevelopment of bands were seen. After this time the membrane was rinsedin distilled water and photographed.

[0088] In an exemplary method, beta-amyloid protein (APP) wasconcentrated from urine specimens of patient having Alzheiemer's by themethod described above and a Western blot performed. The resulting blot,stained by APP-antibody 369, is shown in FIG. 7 of the accompanyingdrawings. Positive results are seen in lane 0, control APP, lanes1,3,4,6,9,10,11 and M from specimens from Alzheimer's patients.

[0089] Lane 3 is control and lane 7 relates to an assay for specimensfrom patients with Parkinson's disease.

Detection of Amyloid Precursor Protein Segments in Alzheimer's Patients

[0090] One hundred ml, or larger, urine specimens, were collected in 50ml tubes, three times, from 10 clinically diagnosed Alzheimer's patientsand 10 healthy individuals of similar age group and sent fresh to thelaboratory. After centrifugation at 1000 g for 10 minutes to removegross debris, the supernatant was transferred to fresh 50 mlpolypropylene centrifuge tubes. One 50 ml aliquot of the specimens wasused and the rest frozen. To each tube, 1 ml buffer was added, mixed andthen 500 μl non-buoyant particulate flock added. Tubes were left on aroller for 30 minutes at room temperature and agitated every 10 minutes.The tubes were then centrifuged at 200 g for 3 minutes and the pelletcollected and supernatant discarded. The pellet of non-buoyantparticulate flock with protein fragments adsorbed was transferred to amicrofuge tube and suspended with another 1 ml buffer and centrifuged.This step was repeated twice. Following concentration of the urine,buffer was removed by centrifugation at 10,000 g for 1 minute and 250 μlsample buffer (3×) was added, mixed and followed by boiling for 3minutes. The supernatant was collected into a fresh tube aftercentrifugation at 10,000 g for 1 minute. This sequence provides anapproximate concentration of 200 times.

Western Blotting

[0091] After boiling, the samples were run on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels. For each run, 20 μlof the 250 μl of the concentrate was loaded. Electrophoresis was carriedout using 10% polyacrylamide gel using BIO-Rad mini-gel apparatus.Secretory amyloid precursor protein C-terminal was used for the control.After the run, the proteins were transferred to PVDF membrane.Unadsorbed sites were then blocked using milk blocking buffer withsod-azide. A first amyloid precursor protein antibody 369 was made up inblocking buffer which was left to incubate for one and a half hours. Themembrane was then washed three times in wash buffer. A second antibody,conjugated to a marker enzyme, (which was also made up in secondaryblocking buffer without sod-azide) was left to incubate for one and ahalf hours and then washed three times in wash buffer without sod-azide.Developing: 1 part of A+1 part of B on membrane for 1 minute. The liquidwas blotted and the membrane exposed for 30 seconds and 5 minutes andthe film developed.

Results

[0092] Western immunoblots prepared from urine concentrates of allAlzheimer's patients showed positive reactivity to the antibody raisedto the amyloid precursor protein segments. Samples include collectionand processing on different days from the same patients. Apart fromquantitative differences, in most cases, two bands of 27 to 30 KD and 7KD were seen. In some patients, there was a third band, just below the27 to 30 KDa band. None of these bands were seen in one patient withParkinson's disease also included in this study. No bands were seen incontrol cases. For comparative purposes, urine specimens from someAlzheimer's disease cases were run in SDS-PAGE gel withoutconcentration. None of the bands were seen in SDS-PAGE gel in theseruns.

Purification of Viral Samples from Water

[0093] Water samples were collected from laboratory tap and also fromthe River Tyne in gallon containers. A 2 to 5% suspension of faeceswhich contained rotavirus was prepared in PBS. One ml of the suspensionwas added into one gallon water sample, mixed by shaking for 2-3minutes. To each container, 10 ml buffer was added, mixed and then thecap of the container was replaced with a ion-exchange filter. The liquidwas poured by gently tilting the container and was discarded. The filterpaper was removed and immersed in 250 μl saturated versene. Followingthe concentration 50 μl of versene was used to prepare the grids by lowspeed centrifugation technique (Narang et al, 1980, Lancet, i,1192-1193). The grids were stained with PTA and examined with anelectron microscope. Rotavirus was found in all water samples with addedfaecal suspension, both in the tap and river concentrated by filtermethod. The filter method can be used to concentrate virus from river,sea and swimming pools water. The number of virus particles seen by anelectron microscope demonstrated that the concentrated water samplescould be used for analysis by Western Blotting.

What is claimed is:
 1. A method of monitoring a liquid for the presenceof disease-modified or associated proteins, comprising the steps of: (a)contacting a sample of said liquid with a solid, non-buoyant particulatematerial having free ionic valencies so as to concentrate saiddisease-modified or associated proteins in said sample; and (b)monitoring the resulting disease-modified or associated proteinsconcentrated on said particulate material.
 2. A method according toclaim 1 , wherein said liquid is a sample of body fluid taken from ananimal.
 3. A method according to claim 2 , wherein said sample of bodyfluid is urine.
 4. A method according to claim 1 , wherein saidparticulate material comprises calcium phosphate in granular form.
 5. Amethod according to claim 1 , wherein said concentrated proteins aremonitored using electron microscopy.
 6. A method according to claim 1 ,wherein said concentrated proteins are monitored using an enzyme linkedimmunosorbent assay (ELISA).
 7. A method according to claim 6 , in whicha first antibody is added to said concentrated proteins so as to permitsaid first antibody to complex with said concentrated proteins.
 8. Amethod according to claim 7 , wherein a second antibody which isconjugated to a marker enzyme is added to said complexed proteins so asto permit said second antibody to complex to said first antibody.
 9. Amethod according to claim 1 , wherein said concentrated proteins areamplified using a polymerase chain reaction and then monitored by arestriction fragment length method.
 10. A method according to claim 1 ,wherein said concentrated proteins are used in a hybridization reactionand then monitored using Western blotting.
 11. A kit for carrying out anELISA reaction, the kit comprising: (a) a solid, non-buoyant particulatematerial having free ionic valencies in a form capable of complexingwith disease-modified or associated proteins present in a sample ofliquid; (b) a blocking buffer capable of complexing with saidparticulate material not complexed with said proteins; (c) a firstantibody material capable of complexing with said complexed proteins;and (d) a further antibody which is capable of complexing with saidfirst antibody.
 12. A kit according to claim 11 , wherein said liquid isa sample of body fluid taken from an animal.
 13. A kit according toclaim 12 , wherein said sample of body fluid is urine.
 14. A kitaccording to claim 11 , wherein said particulate material comprisescalcium phosphate in granular form.
 15. A method for concentratingdisease-modified or associated proteins from a sample of liquid whichcomprises the following steps: (a) collecting and centrifuging saidsample of liquid; (b) collecting the supernatant produced followingcentrifugation of said sample; (c) adding a buffer and a solid,non-buoyant particulate material having free ionic valencies to saidsupernatant; (d) centrifuging the resulting mixture of said buffer, saidparticulate material and said supernatant; (e) collecting saidparticulate material following centrifugation; (f) adding a buffer tosaid particulate material; (g) centrifuging said mixture of said bufferand said particulate material; (h) collecting said particulate material;(i) adding a buffer to said particulate material; (j) centrifuging amixture of said buffer and said particulate material; and (k) collectingsupernatant containing the disease-modified or associated proteins. 16.A method according to claim 15 , wherein said liquid is a sample of bodyfluid taken from an animal.
 17. A method according to claim 16 , whereinsaid sample of body fluid is urine.
 18. A method according to claim 15 ,wherein said particulate material comprises calcium phosphate ingranular form.
 19. A method of monitoring a liquid for the presence ofbiological material selected from the group consisting ofdisease-modified or associated proteins, a fragment thereof, a virus ora fragment thereof, comprising the steps of: (a) providing a sample ofsaid liquid; (b) passing said sample through a solid filter mediumhaving free ionic valencies so as to complex at least one of saidbiological material to said medium; and (c) monitoring at least a partof said complexed biological material, wherein the presence of at leasta part of said biological material is indicative of an association ofsaid liquid with the relevant disease.
 20. A method according to claim19 , wherein said liquid is a sample of body fluid taken from an animal.21. A method according to claim 20 , wherein said sample of body fluidis urine.
 22. A method according to claim 19 , wherein said filtercomprises a gauze fiber material.
 23. A method according to claim 19 ,wherein said filter comprises a cotton fiber material.
 24. A methodaccording to claim 19 , wherein said filter medium comprises asheet-like member with a pore size ranging from 1 to 100 microns.
 25. Amethod according to claim 19 , wherein said complexed biologicalmaterial is monitored using electron microscopy.
 26. A method accordingto claim 19 , wherein said complexed biological material is monitoredusing an enzyme linked immunosorbent assay (ELISA).
 27. A methodaccording to claim 26 , in which a first antibody is added to saidcomplexed biological material so as to permit said first antibody tocomplex with said complexed biological material.
 28. A method accordingto claim 27 , wherein a second antibody which is conjugated to a markerenzyme is added to said complexed biological material so as to permitsaid second antibody to complex to said first antibody.
 29. A methodaccording to claim 19 , wherein said complexed biological material isamplified using a polymerase chain reaction and then monitored by arestriction fragment length method.
 30. A method according to claim 19 ,wherein said complexed biological material is used in a hybridizationreaction and then monitored using Western blotting.
 31. A method ofmonitoring a liquid for the presence of biological material selectedfrom the group consisting of disease-modified or associated proteins, afragment thereof, a virus or a fragment thereof, comprising the stepsof: (a) providing a sample of said liquid; (b) contacting said samplewith a solid, non-buoyant particulate material having free ionicvalencies; (c) centrifuging at least once, said mixture of saidparticulate material and said sample; (d) collecting the supernatant andpassing said supernatant through a solid filter medium having free ionicvalencies so as to complex at least one of said biological material tosaid medium; and (e) monitoring at least a part of said complexedbiological material, wherein the presence of at least a part of saidbiological material is indicative of an association of said liquid withthe relevant disease.
 32. A method according to claim 31 , wherein saidliquid is a sample of body fluid taken from an animal.
 33. A methodaccording to claim 32 , wherein said sample of body fluid is urine. 34.A method according to claim 31 , wherein said particulate materialcomprises calcium phosphate in granular form.
 35. A method according toclaim 31 , wherein said filter comprises a gauze fiber material.
 36. Amethod according to claim 31 , wherein said filter comprises a cottonfiber material.
 37. A method according to claim 31 , wherein said filtermedium comprises a sheet-like member with a pore size ranging from 1 to100 microns.
 38. A method according to claim 31 , wherein said complexedbiological material is monitored using electron microscopy.
 39. A methodaccording to claim 31 , wherein said complexed biological material ismonitored using an enzyme linked immunosorbent assay (ELISA).
 40. Amethod according to claim 39 , in which a first antibody is added tosaid complexed biological material so as to permit said first antibodyto complex with said complexed biological material.
 41. A methodaccording to claim 40 , wherein a second antibody which is conjugated toa marker enzyme is added to said complexed biological material so as topermit said second antibody to complex to said first antibody.
 42. Amethod according to claim 31 , wherein said complexed biologicalmaterial is amplified using a polymerase chain reaction and thenmonitored by a restriction fragment length method.
 43. A methodaccording to claim 31 , wherein said completed biological material isused in a hybridization reaction and then monitored using Westernblotting.